Abstract
BackgroundBecause most transient transformation techniques are inadequate for functional genomics research in roots, we aimed to develop a simple and efficient Agrobacterium-mediated transient transformation system that utilized root absorption for research in flowering Chinese cabbage.ResultsBoth semi-quantitative and fluorescent quantitative RT-PCR confirmed that the target gene BcAMT1;3 was more highly expressed in plants that were infected with the transformed Agrobacterium strain (EHA105-p35S-BcAMT1;3) than in control plants that were infected with the control strain (EHA105-p35S). Furthermore, GUS staining analysis conformed the availability of this transient transformation system. In addition, we found that the highest transformation efficiency was achieved using an Agrobacterium cell density of OD600 = 0.3 for 3–6 h, without hyperosmotic pretreatment, and under these conditions, the peak transformation efficiency was observed at 2 and 4 d after infection.ConclusionsThe transformation method developed by the present study is simple and convenient, since no special equipment is required, and since the method causes no damage, the plants can be used for subsequent experiments.
Highlights
Because most transient transformation techniques are inadequate for functional genomics research in roots, we aimed to develop a simple and efficient Agrobacterium-mediated transient transformation system that utilized root absorption for research in flowering Chinese cabbage
To further examine this transient expression system, the β-glucuronidase (GUS) reporter gene was transformed into wild-type flowering Chinese cabbage roots using the transformation system built above, and GUS activity in roots was detected using stereoscope
Reporter gene GUS was transformed into flowering Chinese cabbage roots, and GUS staining activity detected in roots infected by EHA105-p35SGUS further conformed the availability of this transient transformation system (Fig. 6)
Summary
Because most transient transformation techniques are inadequate for functional genomics research in roots, we aimed to develop a simple and efficient Agrobacterium-mediated transient transformation system that utilized root absorption for research in flowering Chinese cabbage. Functional genomics research often involves transgenic approaches that overexpress or silence genes, and among those approaches, both stable and transient transformation methods are valid for studying the functions and regulatory mechanisms of genes (Parinov and Sundresan 2000; Wroblewski et al 2005). Transient transformation is a more attractive alternative that allows transgenes to be assayed more rapidly and (Janssen and Gardner 1989; Kapila et al 1997), so transient transformation has become the main strategy in functional genomics research and has been increasingly employed for research in various species (Ben-Amar et al 2013; Bhaskar et al 2009; Wu et al 2014; Yang et al 2008). The method has limited applications for research involving roots, since the effects are mainly
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